The invention relates to the field of neural transmission and drug discovery.
U.S. Pat. Nos. 6,090,631 and 5,623,051, incorporated herein by reference, describe a region of the P/Q and N-type calcium ion channels which binds syntaxin and SNAP-25 proteins. This region is located between domains II and III of the xcex11 subunit of these channels. Syntaxin and SNAP-25 are proteins which mediate the docking of presynaptic vesicles required for release of neurotransmitters. As described in these documents, these syntaxin/SNAP-25 binding regions can be used to screen for compounds that will block the binding of these regions to syntaxin and/or SNAP-25, thus inhibiting neuronal transmission.
The conditions surrounding the transmission of signals through neuronal networks clearly affects a variety of physiological responses, including perception of pain, learning, memory, and the like. Modulation of the level of neural transmission and the condition of the presynaptic environment have profound physiological effects, primarily within the nervous system. The primary calcium ion channels that effect neural transmission are these N and P/Q type channels. P/Q type channels have been implicated mostly in the presynaptic terminals of the central nervous system (CNS) while the N type channels appear to dominate in the peripheral nervous system. Thus, P/Q type channels are particularly important in CNS functions such as memory and pain.
Calcium ion channels in general are composed of xcex11 subunits which xcex11 subunits are optionally coupled with additional subunits, but can function alone. According to current terminology, N type channels are comprised of xcex11B subunits while P/Q channels are composed of xcex11A subunits.
It would clearly be desirable to provide compounds that are able to control the presynaptic environment so as to permit a greater control over central nervous system functions, including memory, learning and pain. The present invention provides a mechanism for this control. As will be shown below, a specific region of the xcex11A subunit contains a sequence which binds to the known protein Homer which is described in articles by Xiao, B., et al., Cur. Opinion in Neurobiol. (2000) 10:370-374 and by Tu, J. C., et al., Neuron (1998) 21:717-726. The disclosures of these articles are incorporated herein by reference. As shown in these articles, the Homer protein binds to a multiplicity of targets which are important in signaling and neurotransmission. A consensus sequence which is proline rich is also described.
The invention resides in the identification of a peptide region specific to the P/Q calcium ion channel that is responsible for the cascade of events that results in expression of the gene encoding syntaxin-1A. Thus, use of this peptide in screening assays permits identification of compounds that can be used to regulate the levels of syntaxin-1A available in the presynaptic region and thus modulate such functions as learning, memory and pain.
According to the discovery of the applicants herein, calcium flow through the P/Q ion channel specifically effects the expression of the gene encoding syntaxin in model cell systems and in neuronal cells per se. It has now been found that a specific 4-amino acid sequence approximately 200 amino acids from the C terminus of the P/Q calcium ion channel is the site for interaction of this channel with Homer, a protein known to affect intracellular calcium ion stores, and this interaction is essential for the ability of the P/Q calcium ion channel to effect the expression of the syntaxin-1A encoding gene.
Thus, in one aspect, the invention is directed to a method to identify compounds that affect central nervous system function, such as learning and memory, which method comprises contacting a candidate compound with a peptide that comprises the binding site for Homer which resides proximal to the carboxy terminus of the P/Q calcium ion channel and determining whether said compound binds to said peptide. Compounds that bind to this peptide are identified as compounds that affect central nervous system (CNS) function. This screening assay can be conducted in a straightforward manner by simply assessing the ability of the compound to bind. More commonly, the ability of the candidate compound to inhibit the binding of a ligand known to bind the peptide, including the ability of Homer to so bind, can be used to assess said binding.
In another aspect, the invention is directed to a peptide comprising the sequence of the binding site flanked by additional amino acids, typically those which flank the binding site in the native ion channel, and to antibiotics that are immunospecific for this region. In other aspects, the invention is directed to the compounds so identified and to methods to modulate CNS function using these compounds.